Modified Feulgen staining of DNA with aqueous solution of pinacyanol.

The paper reports on the use of a quinoline dye, pinacyanol, towards staining of acid hydrolysed DNA. The dye as an aqueous solution can be used after treatment of mammalian tissue sections in concentrated phosphoric acid at 5 degrees C for 20 min followed by hydrolysis in 6N HCl at room temperature for 15 min, for staining DNA-aldehyde molecules. It has also been observed that staining of DNA-phosphate groups is also possible in sections treated with cold concentrated phosphoric acid after selective extraction of RNA. Both in situ absorption characteristics of stained nuclei as well as in vitro absorption data of an aqueous solution of the dye have been presented. It has been suggested that staining DNA-aldehydes with pinacyanol, without any primary amino group in its molecules is due to a modified Feulgen reaction.     

Staining of DNA-phosphate groups and DNA-aldehyde molecules with dyes of the azine group

The investigation reports on the use of safranine-SO2 and phenosafranine-SO2, prepared with N HCl or oxalic acid plus potassium metabisulphite, for staining rat liver sections following Feulgen procedure. It has been found that optimum staining of DNA-aldehyde molecules is possible with safranine-SO2 and phenosafranine-SO2, prepared with N HCl and potassium metabisulphite, upto a duration of one week after the preparation of the dye-reagents. Thereafter, staining intensity of the nuclei produced by the dye-reagents is gradually diminished. Staining of acid-hydrolysed sections is also possible with aqueous solutions of these dyes. Moreover, DNA-phosphate groups can also be stained with aqueous solutions of these dyes after selective extraction of RNA with cold phosphoric acid. The in situ absorption spectra of nuclei, stained for DNA-aldehyde molecules with safranine-SO2, phenosafranine-SO2 and aqueous solutions of these dyes, have been presented in this paper. Also presented herein are absorption data of nuclei stained with these dyes after selective extraction of RNA. It has been found that absorption-peaks of nuclei stained differently are different from one another. The implications of these findings have been discussed.     

Azure B staining of DNA-aldehyde molecules of acid-hydrolysed tissue sections

This communication presents a method for the preparation of azure B-SO2 with trichloracetic acid (TCA) and potassium metabisulphite for in situ demonstration of DNA-aldehyde molecules following acid hydrolysis of tissue sections. The shelf-life of such a dye-reagent is slightly more than that of the control, prepared with N HCl and potassium metabisulphite. The slightly increased shelf-life of the experimental dye-reagent has been considered to be due to a somewhat higher pH as compared with that of the control. The in situ absorption characteristics of nuclei stained for DNA-aldehyde molecules with either an aqueous solution of azure B or with TCA-azure B-SO2 show peak-absorption at 600 nm in both cases. This phenomenon has been interpreted as due to the fact that azure B does not contain any primary amino group in its molecules and, therefore, the mode of binding of DNA-aldehydes with this dye is different from that with dyes that contain primary amino group. The implications of some of the findings have been discussed.     

Use of aqueous solutions of two basic dyes for the demonstration of DNA

This paper presents informations as to the ability of aqueous solutions of two basic dyes, such as Dahlia and Victoria blue, belonging to aminotriarylmethane group for the staining of DNA-aldehyde molecules as well as DNA-phosphate groups. It has been found that sections of rat tissues stained with aqueous solutions of these dyes after acid hydrolysis followed by drying between folds of filter paper and treatment in n-butanol for a minute and then by a very brief treatment in a mixture consisting of equal parts of n-butanol and absolute ethanol reveal well-stained nuclei. Tissue sections after acid hydrolysis when stained with aqueous solutions of these dyes and then treated with SO2 water do not reveal any colouration of the nuclei. Since both the dyes are without any primary amino group in their molecules, it has been concluded that the imino group of Dahlia and the tertiary amino group of Victoria blue with cold concentrated phosphoric acid and then stained with any of these dyes also exhibit well-stained nuclei. The absorption spectra of nuclei stained with these dyes for DNA-aldehyde molecules as well as DNA-phosphate groups reveal positions of the peaks of maximum absorption at the same wavelength, which, however, are different in the case of nuclei stained with the two dyes. The implications of these findings have been discussed.     

Detection of DNA in mammalian tissues following removal of RNA with cold phosphoric acid.

The paper deals with the staining of nuclei in mammalian tissue sections with night blue, belonging to diphenylnaphthylmethane group and is devoid of any primary amino group in its molecules but is provided with a secondary amino group and tertiary amino groups. Staining of the DNA-phosphate groups with an aqueous solution of night blue depends upon selective removal of RNA from formalin-fixed mammalian tissues by the use of cold concentrated phosphoric acid for 20 min or 75% phosphoric acid in the cold for 2 h. Moreover, sections from which RNA has been extracted can be hydrolysed in 6N HCl at room temperature for 15 min and then can be stained with the aqueous solution of the dye. Sections of tissues after only acid hydrolysis and staining also reveal very satisfactory staining of the nuclei. Possible mechanism of staining has been suggested.     

Preparation of a new red dye from Janus black and its use in the staining of DNA-aldehyde molecules

This communication presents a method for the preparation of a new red dye from an aqueous solution of Janus black by adding NHC1 and sodium thiosulphate to it. This new red dye when used on acid-hydrolysed tissue sections reveals the presence of red nuclei when sections after staining are dried between folds of filter paper, differentiated in n-butanol, cleared in xylene and mounted. Similarly stained sections when treated with SO2 water show partial leaching of the dye from the nuclei. Tissue sections when treated with cold concentrated phosphoric acid for 20 min and then stained with an aqueous solution of Janus black reveal the presence of orange-red nuclei. The new red dye obtained from Janus black does not respond to treatment under UV rays. The in vitro absorption data of the red dye indicate peaks at 210, 270 and 545 nm. The in situ absorption spectra of nuclei stained with the new red dye following Feulgen procedure reveal the peak of maximum absorption at 560 nm and those of nuclei treated with cold concentrated phosphoric acid and then stained with this red dye reveal peak at 530--540 nm. Some relevant points raised out of this investigation have been discussed.     

Staining of DNA-phosphate groups with a mixture of azure A and acridine yellow.

This paper deals with staining of DNA-phosphate groups with a mixture of an equal parts of aqueous solution of azure A and acridine yellow in a 1:1 proportion and also embodies a study of the absorption properties of the stained nuclei. It also embodies results of sequential staining of nuclei stained first with azure A followed by staining with acridine yellow and vice versa, after extraction of RNA with cold phosphoric acid. The results indicate that the absorption peaks of nuclei differ from those of nuclei stained for DNA-aldehyde molecules with azure A-SO2 or acridine yellow-SO2. The in vitro absorption characteristics of an aqueous solution of azure A and those of an aqueous solution of acridine yellow are also presented herein. The conclusion obtained from this study is that all the phosphate groups of DNA do not take part in the staining process when staining is carried out with azure A or acridine yellow alone after after RNA has been extracted. This is because the nuclei stained with these dyes sequentially show the presence of acridine yellow-DNA and azure A-DNA complex.     

Specific staining of nuclei with aqueous solutions of celestin blue B and gallocyanine

This paper presents methods for specific staining of nuclei with aqueous solutions of celestin blue B and gallocyanine in tissue sections from which RNA has been extracted selectively with concentrated phosphoric acid at 5 degrees C for 20 min or by hydrolysis in 6 N HCl at 28 degrees C for 15 min. It has been found that pH of the freshly prepared celestin blue B dye solution is 3.0 and that of an aqueous solution of gallocyanine is 2.8. These pHs can be lowered to 1.5 with concentrated sulphuric or nitric acid and at this pH staining of the nuclei is possible. But with concentrated sulphuric or nitric acid and at this pH staining of the nuclei is possible. But if the pHs are lowered with concentrated hydrochloric or phosphoric acid, effective use of these dyes is not possible. It has been suggested that some dispersion of the two dyes takes place with concentrated sulphuric or nitric acid which are used to lower the pH. Staining of the nuclei is also possible with an aqueous solution of celestin blue B at pH 3.0 but the same is not possible with gallocyanine at pH 2.8. The absorption spectra of nuclei stained with an aqueous solution of celestin blue B at pH 1.5 and 3.0 are fairly identical, the peak of maximum absorption being at 620 nm. Those of nuclei stained with an aqueous solution of gallocyanine reveal irregular peaks. Possible implications of these findings have been discussed.     

Use of tris-buffer-fortified thionine-SO2 for detection of DNA-aldehyde following Feulgen procedure

This communication presents informations on the use of tris-buffer along with N HCl and potassium metabisulphite for the preparation of thionine-SO2 in staining DNA-aldehyde molecules of acid hydrolysed mammalian liver sections. It has been found that thionine, containing tris-buffer, N HCl and potassium metabisulphite, stains DNA-aldehyde molecules with better result than is possible with the control dye-SO2 reagent that does not contain this buffer. The absorption spectra of nuclei stained with this dye-reagent prepared with tris-buffer have also been presented. Further, it has been found that nuclei stained with the freshly prepared dye-SO2 reagent is bluish-violet, whereas those stained with an old dye-reagent is sky blue in colour. The reason for the slightly enhanced nuclear colouration with the experimental dye-reagent over the control has been considered to be due to slightly increased pH in the former as compared with that of the latter. The mechanism of staining with thionine-SO2 has been considered to be of Feulgen type.     

Cytochemical specificity of acridine red towards RNA and depolymerised DNA.

The paper contains an account of the use of the basic dye, acridine red, of the xanthene group, for staining RNA and depolymerised DNA. The in situ absorption curve of nuclei stained with acridine red indicates a peak of maximum absorption at 560 nm, whereas in vitro absorption characteristics of an aqueous solution of the dye indicate the peak at 550nm. The possibility of using acridine red as a substitute for pyronin, when staining is required for RNA only or in methyl green-acridine red sequence for localising DNA and RNA has been discussed.     

Chromoxane cyanine R. II. Staining of animal tissues by the dye and its iron complexes

The staining properties of chromoxane cyanine R (Colour Index No. 43820, Mordant blue 3; also known as eriochrome cyanine R and solochrome cyanine R) have been studied. Used alone, the dye imparted its red colour to nuclei, cytoplasm and collagen. The dye was extracted by mild alkali but not by acids. Stainability required ionized amino groups in the tissue, and there was also evidence for non-ionic binding of the dye. The colours obtained by staining with mixtures of chromoxane cyanine R and ferric chloride varied with the molar iron: dye ratio and with the pH. Useful staining was seen only between pH 1 and 2. The tissues were coloured either all blue (when Fe: dye was high), or both red and blue (when Fe: dye was low). Lower pH favoured the deposition of red, higher pH the deposition of blue colour. The red was mainly in cytoplasm, blue in nuclei and myelin. Collagen fibres were red or purple, depending on pH and iron: dye ratio. Red colours were differentiated by acid and changed to blue, but not extracted, by mild alkali. The red substance in the stained sections was clearly not the free dye, so it was probably an iron-dye complex. From the effects of various differentiating agents, it was deduced that the red and blue dye-metal complex molecules were bound to the tissue by the dye moiety, not by interposition of iron atoms. Staining by the complexes of iron(III) with chromoxane cyanine R did not involve nucleic acids or other polyanions or the amino groups of proteins. There was evidence for only non-ionic binding of both red and blue complexes. It is suggested that the red colour in sections stained by solutions with low iron: dye ratio is due to a simple carboxylate complex, [Fe₂H(dye)]^?. The blue colour would then result from withdrawal of a proton from the red complex to give [Fe₂(dye)]^2-. The bases that remove the protons may be arginine-rich nucleoproteins of nuclei and phospholipid bases of myelin. Techniques are described for informative simultaneous staining in two colours, and for the selective staining of either nuclei or myelin.     

Hoffman's violet and dahlia as specific stains for animal chromosomes.

The paper deals with staining of the chromosomes of animal testicular materials with two basic dyes, Hoffman's violet and dahlia of the triphenylmethane group, following iodine-dye procedure. The important finding, as presented herein, is that iodinated alcohol after staining can be substituted with various acids, both organic as well as inorganic, all of which act as trapping agent preventing leaching of the dye that binds with the chromosomal DNA. It is clear from this study that RNA is not involved by this process of staining, since treatment of stained sections with cold phosphoric acid at 5 degrees C for 20--25 min and then stained also reveals perfect colouration of the chromosomes without any cytoplasmic staining. The in vitro absorption properties of Hoffman's violet have also been presented herein. The probable mechanism of action of these dyes has been suggested.     

Feulgen type staining with Hoffmann's violet-SO2 under exposure to UV rays.

The paper contains an account of the use of Hoffmann's violet-SO2 under exposure to UV rays during staining acid-hydrolysed DNA of mammalian tissue nuclei. Preparations stained with Hoffmann's violet-SO2 without exposure to UV rays reveal extremely pale violet nuclei but when stained under the influence of UV rays show a considerably faster reaction resulting in a very much deeper staining of the nuclei. Sections after staining with this dye-reagent require n-butanol as differentiating reagent. Possible interpretation for the increase in staining ability of this dye-reagent under exposure to UV rays has been elucidated and the reason for considering the reaction as Feulgen type has been discussed.     

Influence of UV rays on Feulgen-type staining with azure A-SO2 prepared with normal hydrochloric acid and sodium thiosulphate

This communication presents a new method for the preparation of azure A-SO2 for use in Feulgen procedure. The salient feature of this method lies in the fact that azure A-SO2 can be decolourised with normal hydrochloric acid and sodium thiosulphate. The pH of this dye reagent is 2.3 and it is of water colour after filtration. The pH of this dye-reagent is raised to 4.0 with an aqueous solution of sodium hydroxide. Nuclear colouration with this newly developed dye-reagent on acid-hydrolysed DNA of tissue sections becomes fairly satisfactory under the usual laboratory conditions. Staining with this dye-reagent under exposure to UV ray is, however, vastly improved within 5 minutes as compared with the control. Stained sections do withstand treatment in SO2 water without exhibiting any leaching of the dye from the nuclei. Possible mode of action of UV rays in increasing the intensity of staining as well as the speed of reaction has been suggested.     

Cytochemical mechanisms of chromatin fluorescence and staining reactions by thionin solutions

After staining with thionin at low concentration (less than 10(-5) M), the masses of condensed chromatin in chicken erythrocyte nuclei show a red fluorescence under green excitation, which is abolished when they become stained by using concentrations higher than 10(-4) M. The possibility that intercalative binding of a hydrophobic component from the dye solution into DNA accounts for this fluorescence reaction in chromatin is briefly discussed.     

A simple method for the use of gallocyanin-chrome alum as an electron stain*

A method has been elaborated for the demonstration of DNA in the electron microscope. The method uses glutaraldehyde fixed tissue pieces from which RNA has been removed by incubation with RNase. DNA is stained by gallocyanin-chrome alum in the tissue block. Embedding and cutting is done in the usual manner. The method is based on histochemical observations at the light microscope level which show sufficient specificity and a good stoichiometry of the staining reaction.     

A rapid procedure for the staining of chromosomes in fixed animal materials

This paper presents a very simple and reliable procedure for the staining of animal chromosomes employing dyes, such as pyronin G, acridine red, rhodamine B, rhodamine 3GO, belonging to aminoxanthene group and brilliant cresyl blue and methylene violet 3RD, belonging to quinone-imine group. The procedure has been tried on sections of the grasshopper and mouse testes fixed in Dutt's modification of Nawaschin mixture. The method is to deparaffinise sections and then to stain with aqueous solution of these dyes for 2--3 minutes, rinsed with water and dehydrated through grades of ethanol, keeping for 15--30 seconds in each grade with several dips. Preparations are then cleared in xylene and mounted. Stained preparations following this procedure revealed excellent colouration of the chromosomes at all the various stages of mitosis and meiosis, particularly in the case of the grasshopper. Mouse chromosomes stained with these dyes following the same method revealed perfect colouration of the fully condensed chromosomes at all stages of mitosis and meiosis but not of the very early stages, except the sex chromosome. Moreover, grasshopper testis sections when treated with cold concentrated phosphoric acid for varying time-periods and then stained with these dyes also revealed excellent colouration of the chromosomes. The implications of these findings have been discussed.     

Nonmarski differential-interference contrast microscopy in transillumination: its use on unstained or stained sections, smears, and wet mounts, or on fluorochromed sections and cell-culture monolayers

Unstained, lightly stained and conventionally stained microtome tissue sections of two different thicknesses (ca. 4 μm and 1 μm) and also unstained or stained wet mounts of cells were photographed under the microscope using bright-field, positive phase contrast, and Nomarski differential-interference contrast (DIC) in transillumination. The photomicrographs were critically compared. It was found that the density of various stains did not adversely affect the better resolution of the DIC image (as compared to the bright-field image); however, Optical sectioning' of darkly stained objects is not possible. Unstained or stained smears of blood or of epithelial cells of buccal mucosa were examined with DIC in transillumination, then after certain preparatory techniques, the same preparation was examined in the scanning electron microscope, and finally the same areas of the slide were viewed with DIC in epiillumination. Particular attention was given to structures (nuclei and cytoplasm) which appeared in positive or negative relief in the photomicrographs taken by the various techniques. It was concluded that the optically more dense nucleus which always appeared in positive relief by the various methods of examination, was in fact geometrically raised from the surrounding cytoplasm. Acridine orange (AO) stained cell-culture monolayers and H and E stained sections were examined under a fluorescence microscope with DIC optics. By comparing photographs which had been taken with DIC, epi-fluorescence or fluorescence in transillumination, and DIC-fluorescence, it was concluded that the DIC image, which had been superimposed on the fluorescence image, contributed a definite gain in information. Some common errors in the interpretation of the DIC image are discussed; methods of avoiding improper use of equipment are given. The conclusion is drawn that the DIC system is superior to positive and negative phase contrast for the examination of a variety of unstained or stained preparations. Therefore, this method can be used to advantage not only for the examination of unstained preparations, but also on some specimens which have been routinely stained or fluorochromed.     

Studies in fluorescence histochemistry: I. The demonstration of sulphomucins*

Sulphated mucosubstances in sections of coagulant-fixed tissue emit a variable yellow to red fluorescence after being stained with purified coriphosphine at pH 2 to 4. Unfortunately this fluorescence is sometimes indistinguishable from a similarly coloured fluorescence emitted by nuclei, oxidized sulphoproteins and some sialo-mucins, which, however, can be eliminated more or less selectively either by (1) a preliminary Feulgen hydrolysis and condensation with N,N-dimethyl-m-phenylene diamine; or (2) by pretreatment with a solution of ferric alum; or (3) by a previous methylation with methanolic thionyl chloride, followed by reduction with lithium aluminium hydride in hot dioxane and then by saponification (a sequence of reactions which, in theory but not always in practice, should remove all polyanionic groups in the tissue except sulphate groups on sulphated mucopolysaccharides); or (4), best of all, by staining sections in a very dilute solution of thiazol yellow at pH 2 after they have been stained in coriphosphine. The rationale and limitations of these methods are discussed critically.     

Demonstration of DNA with an extra-sensitive Schiff's reagent and a Schiff-type dye-reagent, toluidine blue O-SO2

This paper describes a method for the preparation of Schiff's reagent as well as a Schiff-type dye-reagent, toluidine blue O-SO2 for use in Feulgen procedure. The method involves replacement of the usual N HCl by N H2SO4 and the usual amount of potassium metabisulphite. Following this method of preparation, an extra-sensitive Schiff's reagent is obtained which requires only 4-5 min for optimum nuclear colouration even when staining is performed at 5 degrees C. This Schiff's reagent produces perfect Feulgen staining up to 6 months after preparation. Toluidine blue O-SO2, prepared with N H2SO4 and potassium metabisulphite, also produces perfect Feulgen type staining of the DNA-aldehyde molecules of acid-hydrolysed mammalian tissue sections. Toluidine blue O-SO2 when shaken with activated charcoal and filtered produces very satisfactory result. The shell-life of this dye-reagent is just a week. The suitability of the use of N H2SO4 for the preparation of Schiff's reagent as well as a Schiff-type dye-reagent, toluidine blue O-SO2, has been discussed.